Two-phase system for the production and presentation of foreign antigens in hybrid live vaccines

ABSTRACT

The present invention relates to a genetic engineering process for the optimal production and exposure to the immune system of additional antigen coded for by a live vaccine. The genetic engineering process is based on the use of spontaneous DNA reorganisation in the recombinant live vaccine, such that the recombinant live vaccine spontaneously divides into two subpopulations (A and B), whereby subpopulation A is capable of infecting and acts immunogenically per se as a minimum characteristic and subpopulation B as a minimum characteristic is regenerated by subpopulation A, produces additional antigen and acts immunogenically with respect to said additional antigen. The formation of two subpopulations of the live vaccine ensures, on the one hand, that the infection process necessary for the induction of an immune response takes place and, on the other hand, that the formation of additional antigen by a hybrid live vaccine does not disturb the infection process in order to finally achieve an effective immune response to the additional antigen and the pathogen cross-reacting therewith.

BACKGROUND OF THE INVENTION

The method common in medicine of producing effective protection againstinfectious diseases in human beings and animals is based on theprinciple of active immunisation by using pathogen-specific antigens.There are, for example, prophylactic vaccinations against a number ofdiseases in human beings which are caused by bacteria or viruses.However, vaccinations against fungi and parasites are possible inprinciple too.

The basis for the protective effect of such vaccines is that importantantigens, which in general originate or are derived from the pathogen,are brought into contact with the immune system of human beings oranimals by infection or another suitable administration so that aspecific immune response directed to the administered antigens isinduced. The aim and object of this is to select the administeredantigens in such a way and to present them to the immune system suchthat the induced immune response is directed against the pathogen and asubsequent infection is thus prevented. The induced immune response canbe of either humoral (based on antibodies) or cellular nature or both.

The vaccine containing or forming antigens can be constructed orcomposed in a different way (see Bloom, Nature, 342:115-120, 1989). Asimple method consists in using killed pathogens as the vaccine.Improvements are often achieved by only employing a few isolatedcomponents of the pathogen in the vaccine which represent importantantigens. Moreover, new vaccines often only contain a few well-defined(e.g. purified from the pathogen, or prepared by genetic engineering orotherwise) components which act as an antigen by suitable presentation.Furthermore, every combination of the cited possibilities isconceivable. The common factor of the vaccines is that they consist ofinactivated antigen material.

In contrast to these inactivated vaccines, the further possibility ofimmunising with biologically intact pathogens (so-called live vaccines)and conveying effective protection has long been known. Vaccination withliving viruses (Zanetti, Immunology Today, 8:18-25, 1987) and BCGbacteria (Lotte, Adv. Tuberc. Res. 32:107-193, 1984) fall under thiscategory, for example, as well as oral immunisation with living Ty21aSalmonella (Germanier, J. Infect. Dis. 131:553-558, 1975). The principleof such live vaccines is based on the use of an attenuated (orimmunologically related non-virulent for a certain species) pathogenstrain which is able to cause infection and effective immunologicalprotection against the actual pathogen but is no longer pathogenic perse. Experience in the application of live vaccines lies above all withviruses and bacteria; in principle, however, similar vaccines can alsobe developed on the basis of fungi and parasites. Live vaccines oftenhave advantages over comparable inactivated vaccines because they e.g.convey better immunological protection and are safer and less expensive.

New developments have furthermore shown that it is possible to changelive vaccines by genetic engineering so that they not only present theirown antigens to the immune system but also additional antigens which arederived from a different pathogen species. With such hybrid livevaccines it is possible to achieve immunological protection not onlyagainst the pathogen from which the live vaccine is derived or withwhich it is related but also against pathogens against which the immuneresponse to the additional antigen is directed. Depending on the specieson which the live vaccine is based, one or more additional antigens canbe presented to the immune system and immunological protection can thusbe conveyed. This is already realizable in practice with viral andbacterial live vaccines (see Dougan, J. Gen. Microbiol. 135:1397-1406,1989). In contrast to bacterial or other cellular live vaccines, it ishowever natural for viral live vaccines (as well as for viruses ingeneral) to be able to express their genetic information only afterinfection of a cell. In this case the formation of additional antigen bya viral live vaccine consequently requires the infection of cells of theindividual to be protected. The presentation of additional antigens tothe immune system can on the one hand take place via the infected cellper se or on the other hand via the viral live vaccine which carriesadditional antigen in its packaging.

A considerable problem with the construction and use of such hybrid livevaccines is however the circumstance that the production of additionalantigens often changes the biological properties of the live vaccine anddestabilizes the immunological effect so that the desired immunologicalprotection is not achieved or only to a reduced extent. This can be thecase in particular if the additional antigen is produced in largequantities as would often be required for the induction of a good immuneresponse, and/or if the additional antigen is otherwise toxic for thelive vaccine itself. In other words: The hybrid live vaccine behavesdifferently to the original live vaccine with respect to the course ofan infection and thus with respect to its immunisation potential becauseit produces one or more additional antigens. This also means that,depending on the kind of additional antigens produced by the livevaccine, the infectious properties and the effectiveness of theimmunisation of the live vaccine cannot be inferred in so far as it isat all possible to achieve an effective immune response.

Current experiments to solve this problem in bacterial systems pursuethe goal of controlling the formation of additional antigens in a livevaccine by external influences, i.e. to bring the genes which code forthe formation of additional antigens under the control of an induciblepromoter. The additional antigens of the live vaccine would consequentlyonly be formed in dependence on their external influences or theenvironment. Such external influences can be e.g. a certain substance ora certain temperature (e.g. lac system: De Boer, Proc. Natl. Acad. Sci.USA, 80:21-25, 1983; P₁-system: Remaut, Gene, 15:81-93, 1981). It wouldbe ideal if the external influences required for the formation of theantigens were only to be present where the live vaccine came intocontact with the immune system but not in the course of the infectionprocess so that the infection process necessary for the immune responseis not disturbed. However, this is hardly realizable in practice notonly because the infection process and the initiation of contact withthe immune system are biologically linked but also because theconditions at the site of action of the live vaccine, i.e. in certainareas of the body of the person to be vaccinated, can be purposefullycontrolled with the available means neither with respect to location norwith respect to time.

SUMMARY OF THE INVENTION

It is therefore the object of the present invention to introduce agenetic element into the construction of hybrid live vaccines which, onthe one hand, allows the production of sufficiently large quantities ofadditional antigen at the immunological site of action and, on the otherhand, does not interfere with the course of infection by the hybrid livevaccine and thus the expected immunological protection. This object wasachieved by making the formation of the additional antigens of thehybrid live vaccine dependent on random genetic events which occurrelatively frequently. This principle implies the existence of twosubpopulations/phases which originate from the live vaccine, namely ofthe hybrid live vaccine itself (subpopulation/phase A) which does notproduce any additional antigen and therefore reproduces without itsproperties changing in relation to the original strain and is capable ofa normal course of infection and a normal immune response, and asubpopulation/phase B which may have lost these properties but isconstantly newly regenerated from subpopulation A and releases largequantities of additional antigen at the site of action (see FIG. 1).Subpopulation A therefore has the task of guaranteeing a perfect courseof infection while subpopulation B serves to build up an effectiveimmune response to the additionally formed antigens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. shows a schematic representation of the spontaneous formatiaonof two sub-populations/phases (A and B) of a cellular live vaccine.

FIG. 2. shows an example of a specific DNA deletion in accordance withmodel I of the invention taking into account the role of the controlenzyme.

FIG. 3. shows an example of a specific DNA inversion in accordance withmodel II of the invention.

FIG. 4. shows an example of a genetic element according to the presentinvention for a bacterial live vaccine and the nature of its effect.

FIG. 5. shows an immunoblot (Western) analysis of the formation of CT-Bantigen in hybrid S. typhimurium SL3235 cells. In the figure, 20 ng ofpurified CT-B antigen obtained from Sigma is shown as a reference inlane 1 and 2.5 c 10⁷ cells corresponding to bacterial lysates in lanes 2to 6; lane 2, S. typhirmurium SL3235 cultured at 37° C.; lane 3, S.typhirmurium SL3235 (pTK1) cultured at 28° C.; lane 4, S. typhirmuriumSL3235 (pTK1) cultured at 37° C.; lane 5, S. typhirmurium SL3235 (pYZ17)cultured at 28° C. Position “a” refers to an unspecific cross reactionin the blot, position “b” to the precursor of the CT-B antigen andposition “c” to the mature CT-B antigen. The samples are obtained andthe immunoblot is performed acording to Klauser (EMBO J., 9: 1991-1999,1990).

DETAILED DESCRIPTION OF THE INVENTION

Random events which lead to the formation of subpopulations are naturaland can mostly be traced back to changes in the DNA, so-called(programmed) DNA reorganisations (Borst, Science, 235:658-667, 1987). Inprinciple, all naturally-observed mechanisms of DNA reorganisation canbe used for the task set, provided that they can be reproduced suitablyfrequently in the hybrid live vaccine. The frequency of the formation ofsubpopulation B is preferably 0,1% to 50% per cell and cell generation;in particular cases, the frequency can, however, be higher or lower.Particularly suitable for application in a hybrid live vaccine aresimple mechanisms of DNA reorganisation which occur at specific sites,such as inversion (Craig, Cell, 41:649-650, 1985) or deletion byresolution of transposon cointegration (Grindley, Annual Rev. Biochem.,54:863-896, 1985) of a DNA segment. However, other site-specific DNAreorganisations or such DNA reorganisations which are based onslipped-strand-mispairing (Levinson, Mol. Biol. Evol. 4:203-221, 1987;Stern, Cell, 47:61-71, 1986) seem suitable for the cited object.

It must be the purpose of the cited spontaneously occurring DNAreorganisation in a live vaccine to lead directly or indirectly to theproduction of additional antigen, i.e. to the formation of subpopulationB which produces the antigen. This occurs very simply e.g. bypositioning an expression signal (for example the promoter) of a gene byDNA reorganisation in front of the gene such that said gene changes froma non-expressed to an expressed state. All variants of this principleare possible; but they all have the goal of bringing about a change inthe expression of a gene by DNA reorganisation (see FIG. 2 and FIG. 3).It usually makes sense to switch on genes by DNA reorganisation althoughthe opposite is also possible.

The gene switched on by DNA reorganisation can, on the one hand (model1), be the gene coding for an additional antigen or (if the additionalgene is not a protein but an enzymatic synthesis product, e.g. acarbohydrate) a gene required for the antigen synthesis, or, on theother hand (model II), a gene encoding a protein which controls theexpression of the actual gene coding for the antigen. Model I is thus asystem which by DNA reorganisation directly codes for the synthesis ofthe additional antigen or for an enzyme required for the synthesis whilemodel II represents a system which allows the production of theadditional antigen via a cascade system (see FIG. 2 and FIG. 3). Thecascade system can be realized e.g. in that the gene directly controlledby DNA reorganisation codes for an RNA polymerase which is specific forthe promoter preceding the gene coding for the antigen, or a generegulator which in another specific manner induces the expression of thegene coding for the antigen (e.g. T7 polymerase: Studier, Meth. Enzymol.185:60-89, 1990; lac system: De Boer, Proc. Natl. Acad. Sci. USA,80:21-25, 1983). In this case too, there are all the possibilities ofvariation which nature offers. Whilst model I is on the one hand lesscomplicated, the application of model II has advantages because highlevels of expression can be achieved after one single DNA reorganisationdue to the increasing effect of the cascade. Furthermore, with thismodel several genes which code for different additional antigens withinone hybrid live vaccine can be switched on at the same time.

The realization of the described system is technically particularlysimple in bacterial live vaccines. The genetic element capable of DNAreorganisation can be held in bacteria e.g. on a plasmid or introducedin the genome by means of a phage, a transposon or by homologousrecombination. In the case of the cascade model II, the antigen-encodinggenes, which have special sites for the binding of the gene products ofthe element capable of DNA reorganisation, can be introduced in asimilar manner by using conventional techniques.

Depending on the principle of the underlying DNA reorganisation enzymes(referred to here as “control enzymes”) are necessary which have to beprovided by the cell so that DNA reorganisation can take place at all.In the case of an inversion according to the principle of the phage Muan invertase is necessary, for example, besides cellular factors(Kahmann, Cell, 41:771-780, 1985); in the case of a deletioncorresponding to the mechanism of the resolution of transposoncointegration the enzyme resolvase is necessary, for example, (Reed,Cell, 25:713-719, 1981); the formation of replicative circlescorresponding to the replication of filamentous phages requires interalia the gene 2 product of the phage (Meyer, Nature, 296:828-832, 1982).These enzymes as well as the DNA structure at which they attack(referred to here as “target sites”; e.g.: Mertens, EMBO J.,7:1219-1227, 1988), offer a suitable basis from which to regulate thefrequency with which DNA reorganisation forms the additional antigen andthus to determine the ratio of populations A and B. Manipulation of theexpression of the control enzymes or the target sites by geneticengineering makes it possible to adjust this ratio exactly to thedesired ratio of the populations. However, it is also conceivable tosubject the control enzymes in turn to a superordinate regulatorycontrol in order to change the ratio of populations A and B by externalinfluences (e.g. in dependence on the temperature) (see FIG. 2). Here,too, there are possibilities of variation as desired and given in thestate of molecular biology.

The conventional methods of molecular biology serve for the constructionof genetic elements which undergo DNA reorganisation. Moreover, it isadvantageous to firstly clone such genetic elements in cells which donot synthesise control enzymes in order to keep them stable formanipulation and construction purposes. After preparation such elementscan be inserted into the genome of the live vaccine by the conventionalmethods of molecular biology or can be kept extrachromosomally as aplasmid. The same applies to the genes which code for the controlenzymes as well as with respect to genes indirectly or directly codingfor antigens in the case of model II.

The final hybrid live vaccine is administered in a suitable manner (e.g.by oral dosage or by injection) to the individual to be protected. Theadministered dose of the hybrid live vaccine usually corresponds to thatfor the corresponding non-hybrid live vaccine.

EXAMPLES Example 1

Construction of an invertable DNA element for the production of CT-Bantigen (Vibrio cholerae toxin B—subunit) in Salmonella.

The genetic organisation of the invertable DNA element described here isrepresented in FIG. 4. The element is contained on the plasmid pYZ17 andwas constructed by using the following genes or other nucleotidesequences:

The gin gene is derived from the plasmid pLMugin-X16 (Mertens, EMBO J.3:2415-2421, 1984) and was removed from there firstly as partiallycleaved PvuI fragment which still contained the PvuI cleavage sitecontained in the gin gene itself as the only uncleaved PvuI cleavagesite. This PvuI fragment was provided with EcoRI linkers at its ends andafter cleavage with EcoRI and BamHI isolated as a BamHI/EcoRI fragment.This fragment was linked at the BamHI cleavage site with a syntheticBamHI/ClaI fragment(5′-GGATAAACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTTGGAGATTTTCAACGTG-3′)(SEQ. ID. NO.:1) of the terminator of bacteriophage fd so that arecombinant EcoRI/ClaI fragment was obtained.

The cI857-gene fragment was isolated from the plasmid pRK248 (Bernard,Gene 5:59-76, 1979) by partial cleavage with HindII and subsequentligation with an XhoI linker as an XhoI/Cla fragment.

The EcoRI/ClaI gin fragment was ligated to the cI857 gene fragment viathe ClaI cleavage site and incorporated as an EcoRI/XhoI fragment into aderivative (pYZ13) of the plasmid pBT192 (Zahm, Mol. Gen. Genet.,194:188-194, 1984) which carries an additional (EcoRI/ClaI/XhoI/BglII)polylinker region.

The P₁ promoter was isolated as an XhoII fragment from the plasmidpLC2833 (Remaut, Gene 22:103-113, 1983) and linked via the BclI cleavagesite at both ends with two synthetic BclI/XhoI oligonucleotides(5′-GATCATTTACCGTTTCCTGTAAACCGAGGTTTTGGATAAC-3′) (SEQ. ID. NO.:2) whichcorrespond to the IR sequences (inverted repeats). The resultingfragment consisting of the sequence “IR-P₁-IR” was inserted into thesingular XhoI cleavage of plasmid pfdA4 (Geider, Gene 33:341-349, 1985).

The rrnB T1 transcriptional terminator was obtained as a SalI/XhoIfragment with the nucleotide sequence XhoI 5′9—(SEQ. ID. NO.: 3).

The promoterless CT-B encoding ctxB gene fragment was isolated fromplasmid pTK1 (Klauser, EMBO J. 8:1991-1999, 1990) after cleavage withClaI and subsequent linkage with a BglII linker as a BglII/SalIfragment.

The plasmid pYZ17 was constructed from the above-mentioned components inthe following manner:

The SalI/XhoI T1 terminator fragment was inserted into the singular XhoIcleavage of plasmid pYZ13 and tested for the orientation of theremaining intact XhoI cleavage. Derivatives from pYZ13 which carried theremaining XhoI cleavage site distally to the cI857 gene of pYZ13 werecalled pYZ15. The XhoI IR-P₁-IR fragment was inserted into the remainingXhoI cleavage site of pYZ16 so that the P₁ promoter was orientated tothe cI857 gene. This plasmid (pYZ16) was identified by an asymmetricEcoRI cleavage (FIG. 4). Finally the neo^(r) gene contained in pYZ16 wasreplaced by the ctxB fragment by cleavage with BglII/SalI and subsequentligation to give pYZ17 (FIG. 4). Since pYZ17 possesses a further EcoRIcleavage site downstream from gin besides the EcoRI cleavage site withinthe invertable PI promoter fragment, the orientation of the promoter canbe determined very easily by cleavage with EcoRI, and the inversion ofthe promoters can be followed up very easily. The fractionation of theEcoRI cleavage products in an agarose gel results in two bands ofdefined size of phase A of plasmid pYZ17 corresponding to about 4960 bpand 1840 bp or two bands of defined size of phase B of the same plasmidcorresponding to 4630 bp and 2170 bp.

The construction and analysis of the described plasmids were carried outaccording to the methods described in Sambrook (Molecular Cloning, 2nded., 1989, Cold Spring Harbor Laboratory Press). For explanation of thefunction of the invertable elements on plasmid pYZ17 see the legend toFIG. 4.

Example 2

Oral immunisation of BALB/c mice against CT-B antigen using a two-phaseSalmonella typhimurium live vaccine

Salmonella typhimurium SL3235 (Hoiseth, Nature 291:238-239, 1981) wastransformed according to the MgCl₂/CaCl₂ method (Lederberg, J.Bacteriol., 119:1072-1074, 1974) in separate preparations by plasmidspYZ17 and pTK1. The transformants were frozen in LB medium containing15% glycerin and kept at −75° C. Before the immunisation experimentthese strains were firstly incubated overnight on LB plates containingampicillin (100 mg/ml) at 28° C. Single colonies were then inoculated in10 ml liquid LB medium containing ampicillin (50 mg/ml) and cultivatedovernight at 28° C. 5 ml of these overnight cultures were then added to20 ml LB medium having a temperature of 28° C. and containing ampicillin(50 mg/ml) and incubated for a further 4 hours at 28° C. The cultivatedbacteria were subsequently harvested by centrifugation, washed in 10 mlsaline (0.8% NaCl) and finally suspended in 2 ml saline.

The mice used for immunisation (BALB/c) were given water but no food for16 hours before the vaccination. All the mice were each given an oraldose of 0.2 ml 50% saturated Na₂HCO₃ solution. The mice were thendivided up into groups of 5 and 30 minutes later were each given 0.2 mlof the prepared bacteria suspension administered gastrally with a bluntvaccination cannula: S. typhimurium SL3235 (group 1), S. typhimuriumSL3235/pTK1 (group 2) or S. typhimurium SL3235/pYZ17 (group 3). The oralimmunisation took place at 3 intervals (day 0, 7 and 14) with the samedosage each time. On day 20, serum and intestinal fluid were collected(Manning, FEMS Microbiol. Lett., 28:317-321, 1985). Protease inhibitorwas added to the removed intestinal fluid (Elson, J. Immunol. Meth.,67:101-108, 1984). The samples of the mice were pooled according to thegroups and determined by means of class-specific goat-anti-mouseantibodies in the ELISA test with respect to their specific antibodytiter (Elson, J. Immunol. Meth., 67:101-108, 1984). The results areshown in Table 1.

Discussion

The present invention is illustrated by the following figures incombination with the description and the examples.

FIG. 1 is an idealization of the exponential reproduction of one cell ofthe live vaccine (e.g. of a bacterium) which carries a genetic elementaccording to the present invention which leads to the spontaneousformation of two subpopulations/phases A (open ellipses) and B (closedellipses). While cells remain reproductive in phase A and thereforecapable of infecting, spontaneously formed cells of phase B form largequantities of additional antigen which, on the one hand, leads to theinduction of an additional immune response but, on the other hand, mayinhibit the further reproduction of phase B. In the presented case thefrequency of the formation of phase B is about 20%.

In FIG. 2, X represents the antigen-encoding gene which is separated inphase A from its promoter (P_(X)) by a transcription terminating segment(U) and is therefore not expressed while in phase B the promoter ispositioned directly in front of the gene X so that said gene isexpressed. The deletion of the DNA segment U occurs by site-specificrecombination at the sites IRS (“internal resolution sites” of thetransposon Tn3) by the enzyme resolvase (TnpR of the transposon Tn3;here: closed, downwards pointing triangle). For this it is importantthat the two IRS sites whose nucleotide sequence is defined, areorientated in the same direction and are preferably localised withinsome 100 to 1000 base pairs on the same DNA molecule. The frequency ofthe deletion event can be determined by a number of factors, for exampleby slight sequence changes of one or both IRS sites or by the quantityof resolvase present in the cell. The quantity of this enzyme present inturn depends on the efficiency of the expression of theresolvase-encoding gene R. Provided that this gene in combination withits promoter (P_(R)) possesses an operator (O_(R)) on whichtranscription regulators can bind, it is possible in principle toregulate the frequency of the DNA reorganisation (deletion of segment U)via the expression levels of the control-enzyme-encoding gene (R) byexternal influences, e.g. temperature.

In FIG. 3, the genes X and Y represent antigen-encoding genes which arecontained e.g. on a plasmid of a hybrid bacterial live vaccine in anyarrangement. Promoters (P_(X) and P_(Y)) proceed these genes and arespecific for certain RNA polymerases (e.g. the polymerase of phage T7)which are not formed by the live vaccines/bacterial cells themselves. Inphase A the genes X and Y cannot be expressed since the specificpolymerase is lacking. However, the live vaccine/bacterial cell containsthe gene (S) which codes for this polymerase. The gene S can now beactivated e.g. by DNA inversion whereby the promoter Ps is positioneddirectly in front of the polymerase gene S by the inversion of a segmentso that said gene is expressed. The inversion of the DNA segmentcontaining the promoter is catalysed (e.g. in analogy to the G segmentof the bacteriophage Mu) by an enzyme (gin/invertase) which attacks atwell-defined target sites (IR/inverted repeats) which hold the oppositeorientation on the DNA. The frequency with which the inversion of thefragment carrying the promoter occurs depends on several factors (interalia the quantity of the invertase present in the cell and the structureof the IR sites at which it attacks) and can be individually adjusted orchanged by manipulation by genetic engineering. In this example, thesite-specific inversion of a DNA segment thus initiates a cascade whichfinally effects the activation of the antigen-encoding genes X and Y.

In FIG. 4, the sketches A and B are the two phases of an invertableelement, corresponding to how this can be present in the subpopulationsA or B after introduction into a bacterial strain. The element shownessentially corresponds to model I (direct expression of the antigengene) and possesses a superordinate regulatory control function which isdependent on the temperature as an external influence. In phase A thepromoter P₁ (PL) responsible for the expression of the antigen gene isdirected in the direction of the c1857 gene, which codes for asuperordinate temperature sensitive repressor, and the gin gene, whichcodes for the control enzyme. The consequence of this is that arepressor which is able to function is formed at the permissivetemperature of 28° C. and reduces the transcription from the P₁ promoter(PL). At a higher temperature (e.g. 37° C.) the transcription of the P₁promoter (PL) is increased since the repressor is inactivated at leastpartially under such external influences. The temperature-dependentincrease in the transcription also causes a corresponding increase inthe expression of the following gin gene which as a control enzymecatalyses the inversion of the promoter at the target sites. Thus, thefrequency of the inversion is also increased by a higher temperature andthus the transition of the invertable element in phase B. However, theinversion at a lowered temperature is not completely prevented since thegin gene is always expressed on a low level anyway and, moreover,because the host cell itself which is used as the live vaccine usuallypossesses an active gene similar to gin.

The inversion of the promoter causes less repressor and less controlenzyme to be formed on the one hand and the antigen-encoding gene (CT-B)to be strongly expressed by means of the inverted promoter and due tothe weaker formation of the cI repressor on the other hand.

In the above example, the invertable element is contained in theSalmonella typhimurium live vaccine (SL3235) on the plasmid pYZ17. Sincethis plasmid is present in several copies per bacterial cell, elementsof both phase A and phase B can be present in a single bacterial cell.This results in additional interactions between the plasmids of phases Aand B which cause a fractionation of the live vaccine not only in twosubpopulations but also in the intermediates in between. If this is notdesired, this can be easily taken care of by integration of theinvertable element into the chromosome of the live vaccine or by cloningthe element on a single copy plasmid.

For further functional analysis of the invertable element contained inplasmid pYZ17 on the plasmid pYZ17 see also FIG. 5 and Tab. 1. In thedrawing the abbreviations mean as follows: Eco, EcoRI restriction sitesfor the analysis of the phase state of the invertable element; gin (alsogin in the text), the gene coding for the control enzyme (invertase); T,transcription terminators for the reduction of the gene expression; cI(more precisely referred to in the text as cI857), the gene coding forthe temperature-sensitive regulator (repressor); IR, the target sites ofthe invertase (inverted repeats); PL (also referred to in the text asP₁), the invertable promoter which is regulatable by the cI857repressor; CT-B, the gene (ctxB) coding for the antigen (V. choleraetoxin B-subunit).

Detailed Description of the Invention

FIG. 5 shows evidence of the formation of CT-B antigen by the invertibleelement of plasmid pYZ17 in S. typhimurium SL3235 in comparison withplasmid pTK1 (Klauser, EMBO J., 9:1991-1999, 1990).

It is apparent that S. typhimurium SL3235 (pYZ17) produces very littleCT-B antigen at 28° C. in contrast to at 37° C. It is further evidentthat in contrast thereto the formation of CT-B antigen in the referencestrain S. typhimurium SL3235 (pTK1) is independent of temperature andthat in this strain in relation to S. typhimurium SL3235 (pYZ17), whichwas cultured at 37° C., less CT-B precursor protein is accumulated. Thelatter reflects the formation of little CT-B antigen in subpopulation Aand much CT-B antigen in subpopulation B in S. typhimurium SL3235(pYZ17) cultured at 37° C.: The formation of much CT-B in only a fewcells leads to a hold-up in the conversion from precursor into matureCT-B antigen.

Tab. 1 Humoral immune response after oral immunisation of BALB/c micewith hybrid Salmonella typhimurium SL3235 strains.

The results of the experiment described in Example 2 are represented.The antibody titers stated therein correspond to those dilution levelsof the samples which only just give a positive result in the ELISA test.

TABLE I ELISA titer Intestinal Serum lavage IgG IgA IgG SL3235   20  8 8 SL3235 (pTK1)  640  16 16 SL3235 (pYZ17) 10240 128 64

3 62 base pairs nucleic acid single linear DNA (genomic) not provided 1GGATAAACCG ATACAATTAA AGGCTCCTTT TGGAGCCTTT TTTTTTGGAG ATTTTCAACG 60 TG62 40 base pairs nucleic acid single linear DNA (genomic) not provided 2GATCATTTAC CGTTTCCTGT AAACCGAGGT TTTGGATAAC 40 206 base pairs nucleicacid single linear DNA (genomic) not provided 3 TCGAGGTAGC GAGCTTGAGGCATCAAATAA AACGAAAGGC TCAGTCGAAA GACTGGGCCT 60 TTCGTTTTAT CTGTTGTTTGTCGGTGAACG CTCTCCTGAG TAGGACAAAT CCGCCGGGAG 120 CGGATTTGAA CGTTGCGAAGCAACGGCCCG GAGGGTGGCG GGCAGGACGC CCGCCTTAAA 180 CTGCCACAAG CTCGGTACCGTTAACG 206

What is claimed is:
 1. An immunogenic composition comprising a livingcell or a virus expressing a first immunogenic antigen and furthercomprising recombinant DNA encoding at least one second, immunogenicantigen heterologous to said cell or virus, wherein said at least onesecond antigen is expressed under the control of a DNA reorganizationoccurring in said cell or in a cell infected with said virus, the DNAreorganization resulting in a phase variation in the cell or virus,wherein the DNA reorganization occurs in a subject to which said livingcell or virus is administered.
 2. An immunogenic composition comprisinga population A of a living cell or virus, said population A beinginfectious to a subject and expressing a first antigen and beingimmunogenic as to said first antigen, said living cell or virus furthercomprising at least one recombinant DNA comprising: i) at least onepolynucleotide sequence encoding at least one second antigenheterologous to said cell or virus; ii) a promoter; and iii) at leastone DNA element executing a DNA reorganization; wherein said living cellor virus or said at least one recombinant DNA further comprises: iv) apolynucleotide that encodes an enzyme that effects said DNAreorganization via said at least one DNA element iii); and wherein saidpopulation A forms a subpopulation A′ by DNA reorganization of saidrecombinant DNA, whereby subpopulation A′ expresses said first and atleast one second antigens and is immunogenic as to said first and atleast one second antigens.
 3. The immunogenic composition of claim 2,wherein said promoter ii) is operatively linked to said polynucleotidei) and to said at least one DNA element iii).
 4. The immunogeniccomposition of claim 3, wherein said promoter ii) is one from whichtranscription is effected by a specific polymerase or that is regulatedby a specific gene regulator.
 5. The immunogenic composition of claim 2,wherein said at least one recombinant DNA further comprises: v) aregulatory polynucleotide encoding a specific polymerase or a specificgene regulator; and vi) a second promoter from which transcription iseffected by said specific polymerase or that is regulated by saidspecific gene regulator.
 6. The immunogenic composition of claim 5,wherein said regulatory polynucleotide v) is operatively linked to saidpromoter ii) and is operatively linked to at least one DNA element iii),whereby said specific polymerase or specific gene regulator v) isexpressed upon said DNA reorganization, and said polynucleotide encodingsaid at least one second antigen i) is operatively linked to saidpromoter vi) from which transcription is effected by said specificpolymerase or that is regulated by said specific gene regulator.
 7. Theimmunogenic composition of claim 5, wherein said at least onerecombinant DNA further comprises: vii) a second regulatorypolynucleotide encoding a second specific polymerase or a secondspecific gene regulator, wherein transcription from said promoter ii) iseffected by said second specific polymerase or is regulated by saidsecond specific gene regulator.
 8. The immunogenic composition accordingto claim 1 or 2, wherein said DNA reorganization is a specific DNAinversion.
 9. The immunogenic composition according to claim 1 or 2,wherein said DNA reorganization is a specific deletion.
 10. Theimmunogenic composition according to claim 1 or 2, wherein said DNAreorganization is a specific DNA replication process.
 11. Theimmunogenic composition according to claim 1 or 2, wherein said DNAreorganization occurs due to slipped-strand-mispairing.
 12. Theimmunogenic composition according to claim 2, wherein said DNAreorganization is effected by an enzyme that is expressed under thecontrol of a regulatable promoter.
 13. The immunogenic compositionaccording to claim 1 or 2, wherein said at least one recombinant DNA ispresent in a chromosome of said cell.
 14. The immunogenic compositionaccording to claim 1 or 2, wherein said at least one recombinant DNA ispresent on a plasmid.
 15. The immunogenic composition according to claim1 or 2, wherein said recombinant DNA is present in at least two copies.16. The immunogenic composition according to claim 1 or 2, thatcomprises a bacterium or a eukaryotic cell.
 17. The immunogeniccomposition according to claim 1 or 2, wherein said DNA reorganizationresulting in expression of said at least one second antigen occurs at afrequency ranging from 0.1% to 50% per cell or virus per generation. 18.The immunogenic composition according to claim 1 or 2, that comprises acell of Salmonella typhimurium and wherein said at least one secondantigen is the CTX-B protein of Vibrio cholerae.
 19. An immunogeniccomposition comprising a population A of a living cell or virus, saidpopulation A being infectious to a subject and expressing a firstantigen, said living cell or virus further comprising at least onerecombinant DNA comprising: i) a polynucleotide that encodes an enzymethat catalyzes a DNA inversion; ii) at least one polynucleotide sequenceencoding at least one second antigen heterologous to said cell or virus;iii) a polynucleotide that encodes a specific gene regulator or aspecific polymerase; iv) DNA elements for executing a DNA inversioncatalyzed by said enzyme, said DNA protein binding elements beinglocated between said polynucleotides ii) and iii); and v) a promoterlocated between said DNA elements so that said promoter is operativelylinked to either the polynucleotide ii) or the polynucleotide iii) uponsaid DNA inversion, wherein said promoter is regulated by said specificgene regulator or transcribed by said specific polymerase; wherein saidpopulation A forms a subpopulation A′ by said DNA inversion, wherebysubpopulation A′ expresses said first and at least one second antigensand is immunogenic as to said first and at least one second antigens.20. An immunogenic composition comprising a population A of a livingcell or virus, said population A being infectious to a subject andexpressing a first antigen, said living cell or virus further comprisingat least one recombinant DNA comprising: i) DNA elements executing a DNAreorganization; ii) a promoter; iii) at least one polynucleotidesequence encoding at least one second antigen heterologous to said cellor virus, located between said DNA elements so that said promoter ii) islinked to or unlinked from said at least one polynucleotide sequenceencoding at least one second antigen upon said DNA reorganization; andiv) a polynucleotide that encodes an enzyme that effects said DNAreorganization via said DNA elements i); and wherein said population Aforms a subpopulation A′ by DNA reorganization of said recombinant DNA,whereby subpopulation A′ expresses said first and at least one secondantigens and is immunogenic as to said first and at least one secondantigens.
 21. An immunogenic composition comprising a population A ofsaid living cell or virus, said population A being infectious to asubject and expressing a first antigen, said living cell or virusfurther comprising a recombinant DNA comprising: i) DNA elementsexecuting a DNA reorganization; ii) a first promoter; iii) a regulatorypolynucleotide encoding a specific polymerase or a specific generegulator, located between said DNA protein binding elements so thatsaid promoter ii) is linked to or unlinked from said regulatorypolynucleotide encoding a specific polymerase or a specific generegulator upon said DNA reorganization; iv) a second promoter from whichtranscription is effected by said specific polymerase or that isregulated by said specific gene regulator operatively linked to at leastone polynucleotide sequence encoding at least one second antigenheterologous to said cell or virus; and v) a polynucleotide that encodesan enzyme that effects said DNA reorganization via said DNA elements i);wherein said population A forms a subpopulation A′ by DNA reorganizationof said recombinant DNA, whereby subpopulation A′ expresses said firstand at least one second antigens and is immunogenic as to said first andat least one second antigens.
 22. An immunogenic composition comprisinga population A of a living cell or virus, said population A beinginfectious to a subject and expressing a first antigen, said living cellor virus further comprising at least one recombinant DNA comprising: i)DNA elements executing a DNA reorganization; ii) a promoter; iii) atleast one polynucleotide sequence encoding at least one second antigenheterologous to said cell or virus, wherein said promoter ii) is linkedto or unlinked from said at least one polynucleotide sequence encodingat least one second antigen upon said DNA reorganization; and whereinsaid living cell or virus or said at least one recombinant DNA furthercomprises: iv) a polynucleotide that encodes an enzyme that effects saidDNA reorganization via said DNA elements i); and wherein said populationA forms a subpopulation A′ by DNA reorganization of said recombinantDNA, whereby subpopulation A′ expresses said first and at least onesecond antigens and is immunogenic as to said first and at least onesecond antigens.
 23. An immunogenic composition comprising a populationA of said living cell or virus, said population A being infectious to asubject and expressing a first antigen, said living cell or virusfurther comprising a recombinant DNA comprising: i) DNA elementsexecuting a DNA reorganization; ii) a regulatory polynucleotide encodinga specific polymerase or a specific gene regulator; iii) a firstpromoter located between said DNA protein binding elements so that saidfirst promoter is linked to or unlinked from said regulatorypolynucleotide ii) encoding a specific polymerase or a specific generegulator upon said DNA reorganization; iv) a second promoter from whichtranscription is effected by said specific polymerase or that isregulated by said specific gene regulator operatively linked to at leastone polynucleotide sequence encoding at least a second antigenheterologous to said cell or virus; and v) a polynucleotide that encodesan enzyme that effects said DNA reorganization via said DNA elements i);wherein said population A forms a subpopulation A′ by DNA reorganizationof said recombinant DNA, whereby subpopulation A′ expresses said firstand at least one second antigens and is immunogenic as to said first andat least one second antigens.
 24. The immunogenic composition accordingto any one of the claims 2-6, wherein said population A forms saidsubpopulation A′ upon administration of said immunogenic composition toa subject.
 25. A method for immunizing a subject comprisingadministering the immunogenic composition of claim 1 or 2 to saidsubject in an amount effective to raise an immune response to said atleast one second antigen.